Mix-and-diffuse serial synchrotron crystallography

Abstract

Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzyme at a high level of detail. The success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.

Ultracompact 3D microfluidics for time-resolved structural biology

“…The diffusion coefficient of the iodine x-ray contrast agent in water D = 2 × 10 −5 cm 2 s −1 (ref. 48 ), is~10 times larger than diffusion coefficients of typical enzyme substrates and ligands, such as those used during previous serial crystallography mixing experiments [28][29][30][31] . At 20°C a 50% w/v aqueous sucrose has a viscosity of 8.8 mPa s 49 , compared to water with 1 mPa s. According to the Stokes-relation, D = k B T/6πηr, with the Boltzmann constant k B , temperature T, viscosity η, and particle radius r, the diffusion coefficient for KI in sucrose is about 9 times smaller than that in water.…”

“…However, only a small fraction of biological reactions are naturally induced by light, while most are initiated upon substrate binding. Mix-and-inject time-resolved serial crystallography exploits substrate diffusion into sample crystals for reaction initiation [28][29][30][31] . Generally, microcrystals smaller than 3 µm width are needed for millisecond timeresolution 32 .…”

“…Generally, microcrystals smaller than 3 µm width are needed for millisecond timeresolution 32 . Currently available injector technology has been limited to using T-junctions [28][29][30] or coaxial hydrodynamic flowfocusing geometries 16,31 . Both of these have limited mixing efficiency, which may be improved by augmented 3D flow-refolding features 33 .…”

Die Komplexität der Natur entschlüsseln

“…Sie spielen in der Infektionsforschung eine wichtige Rolle. Einzelpulsbelichtungen in Pump‐Probe‐Schemata sollen den Zusammenhang von biologischen Strukturen und ihrer Dynamik aufklären . Die Abbildung von Zellorganellen mit fein fokussierten Röntgenstrahlen eröffnet einen Zugang zu den Stoffwechselwegen von Krankheitserregern, um neue Medikamente gegen Infektionen entwickeln zu können.…”

Serial protein crystallography in an electron microscope

“…However, during the past few years crystallographic techniques have emerged that are able to exploit nanocrystals for diffraction experiments. Notably, X-ray freeelectron lasers (XFELs) have driven the development of serial crystallography 5-10 , a technique that is also increasingly applied at synchrotron sources [11][12][13][14][15][16][17] . Here, acquiring snapshots in a single orientation from each crystal instead of a rotation series avoids dose accumulation, permitting higher fluences which concomitantly decreases the required diffracting volume.…”